Article
The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units.
Kosan Biosciences Inc., Hayward, CA 94545, USA.
Gene (impact factor:
2.34).
06/2000;
251(1):81-90.
Source: PubMed
-
Citations (0)
- Cited In (27)
-
Article: Roles of fkbN in positive regulation and tcs7 in negative regulation of FK506 biosynthesis in Streptomyces sp. strain KCTC 11604BP.
[show abstract] [hide abstract]
ABSTRACT: FK506 is an important 23-member polyketide macrolide with immunosuppressant activity. Its entire biosynthetic gene cluster was previously cloned from Streptomyces sp. strain KCTC 11604BP, and sequence analysis identified three putative regulatory genes, tcs2, tcs7, and fkbN, which encode proteins with high similarity to the AsnC family transcriptional regulators, LysR-type transcriptional regulators, and LAL family transcriptional regulators, respectively. Overexpression and in-frame deletion of tcs2 did not affect the production of FK506 or co-occurring FK520 compared to results for the wild-type strain, suggesting that tcs2 is not involved in their biosynthesis. fkbN overexpression improved the levels of FK506 and FK520 production by approximately 2.0-fold, and a deletion of fkbN caused the complete loss of FK506 and FK520 production. Although the overexpression of tcs7 decreased the levels of FK506 and FK520 production slightly, a deletion of tcs7 caused 1.9-fold and 1.5-fold increases in FK506 and FK520 production, respectively. Finally, fkbN overexpression in the tcs7 deletion strain resulted in a 4.0-fold (21 mg liter(-1)) increase in FK506 production compared to that by the wild-type strain. This suggests that fkbN encodes a positive regulatory protein essential for FK506/FK520 biosynthesis and that the gene product of tcs7 negatively regulates their biosynthesis, demonstrating the potential of exploiting this information for strain improvement. Semiquantitative reverse transcription-PCR (RT-PCR) analyses of the transcription levels of the FK506 biosynthetic genes in the wild-type and mutant strains proved that most of the FK506 biosynthetic genes are regulated by fkbN in a positive manner and negatively by tcs7.Applied and environmental microbiology 01/2012; 78(7):2249-55. · 3.69 Impact Factor -
Article: FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis.
[show abstract] [hide abstract]
ABSTRACT: BACKGROUND: FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses. RESULTS: Three putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively. CONCLUSIONS: Our results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.BMC Microbiology 10/2012; 12(1):238. · 3.04 Impact Factor -
Article: Improvement of FK506 production in Streptomyces tsukubaensis by genetic enhancement of the supply of unusual polyketide extender units via utilization of two distinct site-specific recombination systems.
[show abstract] [hide abstract]
ABSTRACT: FK506 is a potent immunosuppressant that has a wide range of clinical applications. Its 23-member macrocyclic scaffold, mainly with a polyketide origin, features two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, due to the region-specific incorporation of two unusual extender units derived from methoxymalonyl-acyl carrier protein (ACP) and allylmalonyl-coenzyme A (CoA), respectively. Whether their intracellular formations can be a bottleneck for FK506 production remains elusive. In this study, we report the improvement of FK506 yield in the producing strain Streptomyces tsukubaensis by the duplication of two sets of pathway-specific genes individually encoding the biosyntheses of these two extender units, thereby providing a promising approach to generate high-FK506-producing strains via genetic manipulation. Taking advantage of the fact that S. tsukubaensis is amenable to two actinophage (ΦC31 and VWB) integrase-mediated recombination systems, we genetically enhanced the biosyntheses of methoxymalonyl-ACP and allylmalonyl-CoA, as indicated by transcriptional analysis. Together with the optimization of glucose supplementation, the maximal FK506 titer eventually increased by approximately 150% in comparison with that of the original strain. The strategy of engineering the biosynthesis of unusual extender units described here may be applicable to improving the production of other polyketide or nonribosomal peptide natural products that contain pathway-specific building blocks.Applied and environmental microbiology 05/2012; 78(15):5093-103. · 3.69 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed.
The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual
current impact factor.
Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence
agreement may be applicable.
Keywords
15-methoxy side chains
atypical PKS extender unit ethylmalonyl-CoA
biosynthetic gene cluster
contiguous genes
crotonyl-CoA reductase gene
ethyl side chain
FK506 gene cluster
FK520 biosynthesis
FK520 cluster
FK520 gene cluster
genes flanking
methylmalonyl extender units
novel polyketide extender units
polyketide synthase
putative precursor synthesis genes
rapamycin biosynthesis
Streptomyces hygroscopicus var
Streptomyces sp
three polyketide synthase
unusual PKS extender unit bearing
